In lung microvessel endothelial cells, TNF (i.e., early ~0.5 hr) induces ONOO--mediated nitration of [unreadable]-actin causing dislocation of [unreadable]-catenin from adherence junctions. In our new model of in vivo vascular injury at TNF-24.0 hr, there is a "window" of suppressed vascular permeability within TNF-4.0 hr. Our new preliminary data shows, both in vitro and/or in vivo, that the suppression of (~4.0 hr) TNF-induced increased endothelial permeability is associated with nuclear [unreadable]-catenin translocation and increases in total and membrane VE-cadherin. Our novel preliminary data demonstrates: (1) TNF-24 hr induced barrier dysfunction ex vivo is suppressed by inhibition of glycogen synthetase kinase (GSK)3a/[unreadable] in vivo, (2) T cell factor (TCF)/ lymphoid enhancer factor (LEF)/[unreadable]-catenin dependent promoter activity is increased by TNF in vitro and in vivo, and (3) These responses to TNF are prevented by inhibition of PKC in vivo and PKCa in vitro. This proposal will test the paradigm that TNF-induced (~24.0 hr post- TNF) increase in vascular permeability is suppressed (~4.0 hr post-TNF), at least in part, by PKCa-induced inhibition of GSK3[unreadable] activity. The decrease in GSK3[unreadable] activity causes increased nuclear [unreadable]-catenin. The genomic [unreadable]-catenin-activity increases the expression of VE-cadherin. The increase in VE-cadherin expression promotes zonular-adherence junctions suppressing (i.e., braking) the initial (~0.5 hr post-TNF) increased endothelial protein permeability. The increase in VE-cadherin promotes complex formation with [unreadable]-catenin, the off signal for continuous genomic effects of [unreadable]-catenin. It is proposed that persistent inhibition of GSK3[unreadable] activity will continue to suppress barrier dysfunction. We will map a novel paradigm (i.e., in vitro, in situ and in vivo) for the suppression ("braking") of TNF-induced lung injury which is GSK3[unreadable]-mediated, [unreadable]-catenin-dependent increased VE-cadherin activity. Hypothesis: The hypothesis to be tested is TNF-induced lung injury is suppressed, at least in part by, ?PKCa ? ?GSK3[unreadable] ? ?2-catenin ? ?VE-cadherin in adherence junctions. The Specific Aims are to determine, in vitro and/or in vivo, in endothelium that: (1) TNF causes PKCa-mediated GSK3[unreadable]-inhibition that induces nuclear [unreadable]-catenin translocation (Years 1- 2), (2) The TNF-induced nuclear [unreadable]-catenin translocation causes increased VE-cadherin: (I) promoter activity, (ii) RNA expression, and (iii) protein synthesis (Year 2-3), and (3) The increase in VE-cadherin protein expression suppresses, at least in part, the latter TNF-induced increase in vascular barrier dysfunction (3-4). This proposal will use rat lung microvessel endothelium and lungs isolated from rats treated in vivo. An array of biochemical, genomic, proteonomic and cell biology assays are integrated with lung physiology outcomes. This approach will translate cell-molecular pathways to the mechanisms of lung injury in vivo. PUBLIC HEALTH RELEVANCE: Acute Respiratory Distress Syndrome (ARDS) is a common (150,000 cases/year in the USA) and costly disorder with a mortality rate of ~50%. Sepsis and trauma are major factors predisposing to ARDS. Presently, combat-associated trauma with sepsis is certainly a timely concern due to combat operations in the Iraq and Afghanistan theater. ARDS is mediated, at least in part, by TNF-induced signal transduction pathways. Our strategy in the current proposal is focused on post-receptor, intracellular events which might modify the lung-injury response. Thus, successful completion of the proposed studies may result in progress in treatment and prevention of advanced sepsis/ARDS.